Cyanate has been a useful reagent for studying the mechanism of a number of enzymes. The specific reaction of cyanate with hemoglobin has led to development of an automated extracorporeal system for testing the efficacy of cyanate as a drug for treatment of sickle cell anemia. Recent studies indicate that cyanate can act as a selective inhibitor of protein synthesis in animal tumors. Cyanate administered as a drug or originating naturally from spontaneous breakdown of urea (present at high levels in uremia or chronic diarrhea) has undesirable pharmacological effects. Cyanase is an inducible enzyme present in Escherichia coli which catalyzes the hydrolysis of cyanate. Unusual features of the purified cyanase from E. coli are a requirement for bicarbonate and a stable quaternary structure of 8-10 identical subunits (Mr = 16,350) where pairs of subunits may be covalently joined by disulfide bonds. The catalytic mechanism, biological role, and nature of induction by cyanate of cyanase are unknown. The availability of well characterized cyanase will be of importance if cyanase is to become a useful reagent for therapeutic or analytical purposes. The overall objectives of the proposed study are to: 1) establish the nature of the catalytic mechanism and subunit structure of cyanase (the role of bicarbonate is particularly important, since recent studies suggest that a requirement for bicarbonate as a form of metabolic regulation may be quite common); 2) isolate and characterize a mutant in the cyanase gene, and to use the cyanase deficient mutant (along with growth studies and glutathine and transcarbamoylase deficient mutants) to establish the biological role of cyanase (e.g., does cyanase function to detoxify cyanate originating from carbamoyl phosphate?) and to obtain information about cyanate metabolism and regulation of the cyanase gene (which may be unique); 3) to clone the gene for cyanase; 4) to assess the properties of an immobilized form of cyanase.